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U. E. W. Lange et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2029–2033
This view has been supported by two crystallographic
findings. The two loops described above are tightly
linked by three main chain hydrogen bonds between the
atoms Ala221A O-Gly188 N, Gly226 O-Ala183 N, and
Asn223 N-Leu185 O in trypsin,16 whereas in thrombin
no comparable main chain hydrogen bonds are present
in this region. The temperature factors of these loops
are much higher in thrombin than in trypsin. Both
results are consistent with a higher flexibility of these
loops in thrombin compared to trypsin. Therefore, the
adaptation of Gly219 can be considered as an induced
fit which to this extent is only possible in thrombin, but
not in trypsin, leading to the observed selectivity.
B. M.; Zhang, E.; Tulinsky, A.; Ripka, W. C.; Nutt, R. F. J.
Med. Chem. 1996, 39, 4527.
7. (a) Antonsson, K. T.; Bylund, R.; Gustafsson, N. D.; Nils-
son, N. O. WO9429336, 1994; Chem. Abstr. 1995, 122, 285553.
(b) Bohm, H.-J.; Koser, S.; Mack, H.; Pfeiffer, T.; Seitz, W.;
Hoffken, H. W. Hornberger, W. WO9535309, 1995; Chem.
Abstr., 1996, 124, 233168. (c) Smith, G. F.; Wiley, M. R.;
Schacht, A. L.; Shuman, R. T. WO9523609, 1995, Chem.
Abstr. 1996, 124, 87791. (d) Onshima, M.; Iwase, N.
Sugiyama, S. EP669317, 1995, Chem. Abstr. 1996, 124, 56705.
8. (a) Chlorosulfonyl isocyanate (CSI) mediated introduction
of a nitrile group: Loader, C. E.; Barnett, G. H.; Anderson,
H. J. Can. J. Chem. 1982, 60, 383. (b) Formylation with a,a-
dichloromethyl methyl ether: Petrov, O. I.; Kalcheva, V. B.;
Antonova, A. T. Collect. Czech. Chem. Commun. 1997, 62, 494.
9. Jendralla, H.; Seuring, B.; Herchen, J.; Kulitzscher, B.; Wun-
ner, J.; Stuber, W.; Koschinsky, R. Tetrahedron 1995, 51, 12047.
10. (a) Thrombin assay: 1. Reagents: Thrombin from human
plasma (No. T-8885, Sigma, Deisenhofen, Germany). 2. Sub-
strate: H-d-Phe-Pip-Arg-pNA.2HCl (S-2238, Chromogenix,
Molndahl, Sweden).
In summary, an approach towards selective thrombin
inhibitors of the d-Phe-Pro-Arg type exploiting the dif-
ferences of the S1 pockets of thrombin and trypsin has
been described. Although the observed selectivity is
moderate the effect it is based on could potentially be
used in the molecular modeling assisted design of highly
selective thrombin inhibitors. Furthermore, the struc-
tural information disclosed herein provides the basis for
future investigations in fields like molecular dynamics.
It is expected that such studies will give a deeper insight
into the underlying mechanism of the observed back-
bone flexibility in enzymes like thrombin and may also
help to refine today’s modeling tools,17 which altogether
would have failed to predict the observed selectivity and
subtle changes in the protein structure induced by the
inhibitor.
(b) Trypsin assay: 1. Reagents: Trypsin from bovine pan-
creas (No T-8003, Sigma, Deisenhofen, Germany). 2. Substrate
1
2
.
R -Ile-Glu-(O-OR )-Gly-Arg-pNA HCl (S-2222, Chromogenix,
Molndahl, Sweden).
Buffer (for both assays): Tris 50 mmol/L, NaCl 154 mmol/
L, pH 8.0.
Experimental procedure (for both assays): The chromogenic
assay for the determination of thrombin activity (trypsin
activity, respectively) was performed in 96-well microplates
(No. 650101, Greiner, Nurtingen, Germany). 10 mL of sub-
stance solution in DMSO were added to 250 mL of buffer con-
taining thrombin at a final concentration of 0.1 NIH-units/mL
(or 0.1 mg/L trypsin) and incubated for 5 min at 20–28 ꢁC.
The assay was started by addition of 50 mL of substrate solu-
tion in buffer (final concentration 100 mmol/L) and stopped
after a 5 min incubation period at 20–28 ꢁC by addition of 50
mL of citric acid (35%). The absorbance in each well was
measured against blank at 405/630 nm in a double beam
microplate photometer (MR5000, Dynatech, Denkendorf,
Germany).
The concentration effect curves and the IC50 values were
calculated from data points by multiple iterations using a
nonlinear sigmoidal least squares regression fit algorithm.
11. Baucke, D.; Lange, U. E. W.; Hoffken, W.; Hornberger,
W.; Mack, H.; Seitz, W. Unpublished results.
12. Gustafsson, D.; Antonsson, T.; Bylund, R.; Eriksson, U.;
Gyzander, E.; Nilsson, I.; Elg, M.; Mattsson, C.; Deinum, J.;
Pehrsson, S.; Karlsson, O.; Nilsson, A.; Sorensen, H. Thromb.
Haemost. 1998, 79, 110. Melagatran: Ki (thrombin)=2 nM, Ki
(trypsin)=4 nM.
Acknowledgements
We thank H.-J. Helfrich and P. Scarano for chemical
synthesis, A. Freytag for the in vitro assays and P. Reis
and W. Houy for carrying out the soaking experiments
and recording the X-ray structures.
References and Notes
1. Hemostasis and Thrombosis: Basic Principles and Clinical
Practice, 3rd ed.; Colman, R. W., Hirsch, J., Marder, V. J.,
Salzman, E. W.; Eds.; J. B. Lippincott: Philadelphia, 1994.
2. (a) Coburn, C. A. Exp. Opin. Ther. Patents 2001, 11, 721.
(b) Steinmetzer, T.; Hauptmann, J.; Sturzebecher, J. Exp.
Opin. Invest. Drugs 2001, 10, 845. (c) Rewinkel, J. B. M.;
Adang, A. E. P. Curr. Pharm. Des 1999, 5, 1043. (d) Wiley,
M. R.; Fisher, M. J. Exp. Opin. Ther. Patents 1997, 7, 1265.
3. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun.
1967, 27, 157.
13. Wiley, R. M.; Chirgadze, N. Y.; Clawson, D. K.; Craft,
T. J.; Gifford-Moore, D. S.; Jones, N. D.; Olkowski, J. L.;
Weir, L. C.; Smith, G. F. Bioorg. Med. Chem. Lett. 1996, 6,
2387. The authors report a 130-fold selectivity of thrombin
inhibitor d-Phe-Pro-p-aminomethylbenzamidine versus tryp-
sin based on Kass values. The IC50 values we obtained for the
above compound did not show the same selectivity: 3.45ꢂ10ꢀ8
M (thrombin), 9.91ꢂ10ꢀ8 M (trypsin).
4. Bode, W.; Mayr, I.; Baumann, U.; Huber, R.; Stone, S.;
Hofsteenge, J. EMBO J. 1989, 8, 3467.
14. Crystals of human thrombin with an exosite decapeptide
(Bernard, H. E. J.; Hoffken, H. W.; Hornberger, W.; Rubsa-
men, K.; Schmied, B. Pept.: Chem., Struct. Biol., Proc. Am.
Pept. Symp., 13th Meeting 1993; Hodges, R. S.; Smith, J. A.,
Eds.; ESCOM: Leiden, Netherlands, 1994, pp 592–594.) were
grown according to Skrzypczak-Jankun, E.; Carperos, V. E.;
Ravichandran, K. G.; Tulinsky, A.; Westbrook, M.; Mar-
aganore, J. M. J. Mol. Biol., 1991, 221, 1379.
5. (a) Baucke, D.; Lange, U.; Mack, H.; Seitz, W.; Zierke, T.;
Hoffken, H.-W.; Hornberger, W. WO9806741, 1998; Chem.
Abstr. 1998, 128, 192940. (b) Lange, U. E. W.; Baucke, D.;
Hornberger, W.; Mack, H.; Seitz, S.; Hoffken, H. W. manu-
script in preparation. (c) Lange, U. E. W.; Zechel, C. Bioorg.
Med. Chem. Lett. 2002, 12, 1571.
6. For similar approaches towards selective thrombin inhibi-
tors in other structural classes see: Levy, O. E.; Semple, J. E.;
Lim, M. L.; Reiner, J.; Rote, W. E.; Dempsey, E.; Richard,
Compound 12 was soaked into the preformed thrombin
crystals. Diffraction data were collected on a conventional